The present invention relates generally to the field of pharmaceutical and diagnostic compositions useful in the diagnosis, treatment and prophylaxis of Lyme borreliosis. More specifically, the invention provides an isolated natural surface antigen of Borellia and antibodies thereto for use in the diagnosis, treatment or prevention of Lyme disease.
Borrelia burgdorferi (sensu lato) is a generic term which encompasses several Borrelia species associated with, and believed to be, the causative agent of Lyme borreliosis (Lyme disease): B. burgdorferi sensu stricto, B. garinii, and B. afzelii. This disease is transmitted by the bite of various species of Ixodes ticks carrying the spirochete. The main reservoir of the infection in the United States is the white footed mouse, Peromyscus leucopus, and the infection can be transmitted to many mammalian species including dogs, cats, and man [J. G. Donahue, et al, Am. J. Trop. Med. Hyg., 36:92-96 (1987); R. T. Green, et al, J. Clin. Micro., 26:648-653 (1988)]. Despite the presence of an active immune response, the disease persists for years in patients. Such persistence is postulated to be the result, at least in part, of antigenic variation in the bacterial proteins [J. R. Zhang et al, Cell, 89:275-285 (1997)].
The diagnosis of Lyme disease in humans and animals has been compromised by the lack of definitive serology leading to rapid and accurate testing. Current diagnostic tests suffer from low sensitivity and specificity, as illustrated by a recent survey of diagnostic laboratories"" performance issued by the Wisconsin State Laboratory of Hygiene [L. Bakken et al, J. Clin. Microbiol., 35:537 (1997)]. A simple, sensitive and specific diagnostic composition and method for early detection of Lyme disease is needed in the art.
Publications relating to proteins and polypeptides of Borrelia burgdorferi have suggested their use as diagnostic or pharmaceutical agents. Such proteins and polypeptides include outer surface proteins A and B (OspA and OspB), flagellin, and other proteins designated P21, P39, P66, and P83 according to their estimated molecular weights [A. G. Barbour et al, Infect. Immun 45:94-100 (1984); W. J. Simpson et al, J. Clin. Microbiol., 28 1329-1337 (1990); K. Hansen et al, Infect Immun., 56:2047-2053 (1988); K. Hansen et al, Infect. J. Clin. Microbiol., 26 338-346 (1988); B. Wilske et al, Zentral, Bakteriol. Parasitenkd. Infektionshkr. Hyg. Abt. 1 Orig. Reihe. A. 263:92-102 (1986); D. W. Dorward et al, J. Clin. Microbiol., 29:1162-1170(1991); published NTIS U.S. patent application No. 485,551; European patent application No. 465,204, published Jan. 8, 1992; International Patent Application No. PCT/US91/01500, published Sep. 19, 1991; International Patent Application No. PCT/EP90/02282, published Jul. 11, 1991; International Patent Application No. PCT/DK89/00248, published May 3, 1990; International patent application No. WO92/00055, published Jan. 9, 1992].
A preferred protein candidate for a vaccine is OspA [M. Philipp et al, J. Spirochetal and Tick-borne Diseases, 3:67-79 (1996)]. The expression of OspA is either abrogated or down-regulated when the spirochetes are en route from the tick""s midgut to the salivary glands, as blood feeding is taking place [A. DeSilva et al, J Exp. Med. 183:271-275 (1996)]. This phenomenon generates potential problems that may diminish the OspA vaccine""s efficacy. Spirochetal attrition may occur only within the tick midgut [A. DeSilva et al, cited above] and not upon infection of the vertebrate host. Because the saliva of Ixodes scapularis contains a decomplementing factor [T. Mather et al, xe2x80x9cIxodes saliva: vector competence for Borrelia burgdorferi and potential vaccine strategiesxe2x80x9d, in VII International Congress on Lyme Borreliosis, San Francisco, Calif. (1996)], spirochetal attrition within the tick""s midgut might occur via a mechanism involving only antibody and not complement.
Although the mode of action of antibody-dependent killing is not fully understood, it appears to be a less efficient mechanism of killing than that mediated by antibody and complement, acting together [M. Sole et al, Infect. Immunol., 66:2540-2546 (1998)]. Thus, it may permit evasion from the midgut of those spirochetes that have a low surface density of OspA. Indeed, although OspA is an abundant B. burgdorferi protein, only a minor fraction of OspA molecules is exposed on the outer surface of the spirochete [D. Cox et al, Proc. Natl. Acad. Sci., 93:7973-7978 (1996)]. Thus, small variations in the absolute number of OspA surface molecules may cause significant differences in the spirochete attrition rate and make it possible for a fraction of the resident spirochetes to abscond to the salivary glands.
OspA escape mutants will readily avoid killing altogether, and if they are infectious to the vertebrate host, they will contribute to diminish the OspA vaccine efficacy even further. In clonal populations of B. burgdorferi which are allowed to grow in vitro, the prevalence of mutants that resist killing by anti-OspA antibody ranges between 10xe2x88x925 and 10xe2x88x922 [A. Sadziene et al, J. Exp. Med, 176:799-809 (1992)]. If such frequencies are reproduced in a feeding nymph, in which spirochete numbers may reach a mean of 7,848 within 15 hours of attachment [A. DeSilva et al, Am. J. Trop. Med. Hyg., 53:397-404 (1995)] then several mutants may be present in a single tick. Typical mutant phenotypes include those that express neither OspA nor OspB and, frequently, expressors of a chimeric molecule composed of an N-terminal fragment of OspA fused to a C-terminal fragment of OspB. These deletion mutants have been found in multiple strains of B. burgdorferi [P. Rosa et al, Mol. Microbiol., 6:3031-3040 (1992)] and in several tick isolates from California [T. Schwan et al, J. Clin. Microbiol., 31:3096-3108 (1993)]. Chimeric (deletion) mutants, which are able to resist killing with anti-OspA antibody alone, are killed by the combined action of antibody and complement. Since complement appears to be nonfunctional within the tick [T. Mather et al, cited above], and OspA, and probably its chimeric form as well, are not expressed within the vertebrate host shortly after infection, chimeric OspA escape mutants could infect the vertebrate host. Further, it is estimated that up to 2% of Ixodid ticks are only partially fed and are therefore still questing [Y. Lobet, personal communication]. If such ticks have taken their incomplete meal from a B. burgdorferi-infected host, they will have spirochetes in their salivary glands which do not express OspA and which will readily infect an OspA-vaccinated host.
No booster effect has been observed [M. Philipp et al, cited above] nor should one be expected upon spirochetal challenge of an OspA-vaccinated host. Hence, the OspA vaccine may require repeated administrations to maintain effective antibody titers.
There is a thus a need in the art for additional, and improved, methods and compositions for prevention of Lyme disease in humans and animals, and for treatment thereof.
The present invention satisfies the need in the art by providing methods and compositions for prevention of Lyme Disease based on spirochetal antigens that are expressed by the spirochete during its residence in the vertebrate host. Such methods and compositions may also be employed for the treatment of Lyme Disease.
In one aspect, the invention provides an isolated Borrelia burgdorferi sensu lato surface antigen which is expressed in vivo by the spirochete in the vertebrate host. The antigen, referred to herein as P39.5, is further characterized as having a relative molecular mass of 39.5 kDa. Fragments of this antigen are also useful in the compositions and methods of this invention.
In another aspect, the invention provides novel Borrelia cassette string polypeptides/proteins.
In yet another aspect, the invention provides nucleic acid sequences encoding P39.5 or a fragment thereof, such as P7-1, as well as nucleic acid sequences encoding other Borrelia cassette string proteins or fragments thereof. These nucleic acid sequences include sequences which hybridizes to the above sequences under stringent conditions, allelic variants thereof and deletion mutants thereof.
In another aspect, the invention provides novel proteins comprising fragments of the P39.5 protein sequence or cassette string protein sequences described above, optionally fused to, or mixed with, a second selected polypeptide or protein, which may be a protein of up to about 90% identity in its amino acid sequence with that of P39.5, or other Borrelia antigens, such as OspA, and proteins or polypeptides derived from other microorganisms.
In still another aspect, the invention provides novel protein compositions comprising the protein sequences of the antigen(s) described above, or fragments thereof, optionally mixed with a second selected polypeptide or protein, which may be a protein of up to about 90% identity in its amino acid sequence with that of P39.5, or other Borrelia antigens, such as OspA, and proteins or polypeptides derived from other microorganisms.
In still a further aspect, the invention provides a method for recombinantly producing the above-described P39.5 protein, fragments thereof or fusion proteins containing such fragments, by expressing a DNA sequence encoding the protein, fragment or fusion protein in a selected host cell, and isolating the protein therefrom. Host cells transformed with such DNA sequences are also provided herein.
In still another aspect, the invention provides an isolated antibody directed against the above-described P39.5 antigen, fragments thereof, cassette string proteins or fragments, or a fusion protein containing such fragments. These antibodies may be polyclonal. The present invention also provides a method for producing such antibodies comprising immunizing a human or an animal with an isolated antigen as described above, or with one or a mixture of more than one fragment(s) thereof, or fusion protein containing one or more fragment(s) of this invention. Other types of antibodies may be prepared from such immunized animals, e.g., recombinant, monoclonal, chimeric, humanized, etc.
In another aspect, the invention provides a therapeutic composition and methods for treating humans and/or animals with Lyme disease. The therapeutic composition contains an antibody, or protein, or fragment as described above and a suitable pharmaceutical carrier.
In a further aspect, the invention provides vaccine compositions and methods of vaccinating a human or animal patient against Lyme Disease by use of these above-described compositions. The compositions contain an effective amount of at least one Borrelia antigen of this invention, e.g., an antigen that is expressed in vitro by Borrelia spirochetes, said antigen having a relative molecular mass of 39,500 daltons, or antigenic fragment(s) thereof, or a fusion protein containing such a fragment, or cassette string protein(s), and a pharmaceutically acceptable carrier. The vaccine composition may contain the P39.5 protein, fragments thereof, fusion proteins or mixtures of proteins as described above.
In yet a further aspect, the invention provides vaccine compositions and methods of vaccinating a human or animal patient against Lyme Disease by use of nucleic acid compositions, e.g., DNA vaccines. The compositions contain an effective amount of a DNA sequence encoding at least one Borrelia antigen of this invention or antigenic fragment(s) thereof, cassette string antigen(s), or a fusion protein containing such a fragment, and an optional pharmaceutically acceptable carrier.
In yet a further aspect, the invention provides a method for diagnosing Lyme borreliosis in a human or animal. This method includes the steps of incubating an antigen or antibody of this invention, preferably conventionally labeled for detection, with a sample of biological fluids from a human or an animal to be diagnosed. In the presence of B. burgdorferi infection of the human or animal patient, an antigen-antibody complex is formed. Subsequently the reaction mixture is analyzed to determine the presence or absence of these antigen-antibody complexes. In a further embodiment, the diagnostic assay employs DNA sequences, preferably anti-sense sequences, sense sequences, of the antigen or fragments thereof, and diagnoses infection by the presence of sequences in a biological fluid from the patient that hybridizes thereto. Other conventional assay formats may be employed using reagents identified by this invention.
In another aspect the invention provides a kit for diagnosing infection with B. burgdorferi in a human or an animal patient sample which contains at least one antibody capable of binding at least one antigen of this invention of antigenic fragment(s) thereof, or a DNA sequence encoding one or more antigen(s) of this invention or an anti-sense sequence thereof. The antibodies and sequences may be optionally labeled for detection, or a detection system may be included in the kit.
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.